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PLoS One. 2014 Dec 1;9(12):e114043. doi: 10.1371/journal.pone.0114043. eCollection 2014.

Apical localization of inositol 1,4,5-trisphosphate receptors is independent of extended synaptotagmins in hepatocytes.

Author information

1
Section of Digestive Diseases, Department of Internal Medicine, Yale University, New Haven, Connecticut, United States of America.
2
Department of Cell Biology, Yale University, New Haven, Connecticut, United States of America.
3
Department of Cell Biology, Yale University, New Haven, Connecticut, United States of America; Department of Biomedical Engineering, Yale University, New Haven, Connecticut, United States of America; Kavli Institute for Neuroscience, Yale University, New Haven, Connecticut, United States of America.

Abstract

Extended synaptotagmins (E-Syts) are a recently identified family of proteins that tether the endoplasmic reticulum (ER) to the plasma membrane (PM) in part by conferring regulation of cytosolic calcium (Ca2+) at these contact sites (Cell, 2013). However, the mechanism by which E-Syts link this tethering to Ca2+ signaling is unknown. Ca2+ waves in polarized epithelia are initiated by inositol 1,4,5-trisphosphate receptors (InsP3Rs), and these waves begin in the apical region because InsP3Rs are targeted to the ER adjacent to the apical membrane. In this study we investigated whether E-Syts are responsible for this targeting. Primary rat hepatocytes were used as a model system, because a single InsP3R isoform (InsP3R-II) is tethered to the peri-apical ER in these cells. Additionally, it has been established in hepatocytes that the apical localization of InsP3Rs is responsible for Ca2+ waves and secretion and is disrupted in disease states in which secretion is impaired. We found that rat hepatocytes express two of the three identified E-Syts (E-Syt1 and E-Syt2). Individual or simultaneous siRNA knockdown of these proteins did not alter InsP3R-II expression levels, apical localization or average InsP3R-II cluster size. Moreover, apical secretion of the organic anion 5-chloromethylfluorescein diacetate (CMFDA) was not changed in cells lacking E-Syts but was reduced in cells in which cytosolic Ca2+ was buffered. These data provide evidence that E-Syts do not participate in the targeting of InsP3Rs to the apical region. Identifying tethers that bring InsP3Rs to the apical region remains an important question, since mis-targeting of InsP3Rs leads to impaired secretory activity.

PMID:
25437447
PMCID:
PMC4250053
DOI:
10.1371/journal.pone.0114043
[Indexed for MEDLINE]
Free PMC Article

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